Phage display is a powerful technology to identify peptide sequence motifs that target a particular tissue or cell type in the body (Arap et al., 2002; Kolonin et al., 2004; Sidhu, 2001). Coupling such peptides to drugs and genes would enable their targeted delivery to specific cells and tissues in vitro and in vivo (Arap et al., 2002; Petty et al., 2007; Sergeeva et al., 2006; White et al., 2004). A commonly used platform is the combinatorial filamentous M13 phage library that displays short random peptides fused to a minor coat protein (pIII) (Sidhu, 2001), which can be used to isolate specific cell type-binding peptides by a procedure called biopanning (Petty et al., 2007). In the present invention, the technology has been applied to identify peptide motifs that recognize, and are specifically taken up by, neurons in the dorsal root ganglion (DRG) in mice as an exemplary model for humans. DRG neurons are target cells for the treatment of diseases of the peripheral sensory nervous system (Sghirlanzoni et al., 2005), in certain embodiments of the invention For instance, neuropathic pain is a common symptom in various disorders, including metabolic abnormalities, malignancies, physical injuries, toxins and poisons, and hereditary diseases (Sghirlanzoni et al., 2005), and is the cause of much morbidity and misery. Although various pharmaceutical agents, anesthetics, surgical operations, or procedures such as transcutaneous electrical nerve stimulation, have been used to treat the symptoms of neuropathic pain (Mendell and Sahenk, 2003), such palliative treatments are mostly non-targeted and of limited efficacy (Mendell and Sahenk, 2003). DRG neurons are the primary afferent neurons and can be classified into two broad groups: large and small neurons. Large neurons are thought to be involved mainly in proprioception, while most small neurons are involved in nociception (Zhang and Bao, 2006). Both neuronal populations have also been subclassified by biochemical and histological methods, such as lineage tracing or immunostaining of different markers (neurotransmitters, cell surface carbohydrates) (Dodd and Jes sell, 1985; Kusunoki et al., 1991; Marmigere and Ernfors, 2007; Price and Flores, 2007). The present invention provides novel therapeutics and treatment methods for disease processes affecting the peripheral sensory nervous system (Goss, 2007).
Dysfunction of neurons in dorsal root ganglion (DRG) occurs in a variety of sensory neuronopathies (Sghirlanzoni et al., 2005; Kuntzer et al., 2004), including hereditary (Swanson et al., 1965), autoimmune (Malinow et al., 1986), nutritional (Montpetit et al., 1988), metabolic (Yasuda et al., 2003) and neoplastic diseases (Wanschitz et al., 1997). DRG neuronal disorders are associated with neuropathic pain, loss of sensation and sensory ataxia (Sghirlanzoni et al., 2005; Kuntzer et al., 2004). Delivery of neurotrophic factors can minimize neuronal damage in the DRG (Chattopadhyay et al., 2005; Wang et al., 2005; Pezet et al., 2006). However, neurotrophic polypeptides are susceptible to proteolytic degeneration and their therapeutic effects are short-lived; direct delivery of therapeutic genes proves to be more effective (Xu et al., 2003). Herpes simplex virus (Chattopadhyay et al., 2005) and poliovirus (Jackson et al., 2003) injected through subcutaneous or intramuscular routes are taken up by endocytosis at nerve terminals and travel via axonal transport to somas of DRG neurons. While the approach works in normal functioning nerves, the efficacy of such a strategy is greatly compromised under disease conditions. To circumvent this problem, direct injection into DRG neurons by microneurosurgery can be accomplished by the removal of a piece of vertebra to gain access to the DRG (Xu et al., 2003; Glatzel et al., 2000). Such a maneuver is, however, not practical for repeated injections, which is likely required for chronic disorders. In contrast, intrathecal (IT) injection is a far less invasive procedure that is used routinely in clinical practice (Wang et al., 2005). Nevertheless, DRGs are ensheathed in the dura and bathed in cerebrospinal fluid (CSF). Gene delivery vectors could disperse and be taken up elsewhere leading to complications, including meningitis (Driesse et al., 2000). To optimize the chance for successful DRG neuron delivery while minimizing undesired off-target effects, targeted gene delivery would be an ideal strategy for delivering genes to DRG neurons (Waehler et al., 2007).
Adenovirus (Ad) is an efficient gene transfer system in vitro as well as in vivo because of its capacity to infect both quiescent and proliferating cells, a broad spectrum of tissue transduction susceptibility and relative ease with which the vector can be produced in high titer. Helper-dependent adenoviral vector (HDAd) is the most advanced Ad, which is devoid of all viral coding sequences. The lack of potentially harmful Ad viral gene expression is associated with markedly reduced toxicity. HDAd-mediated in vivo gene delivery in rodents and nonhuman primates has been found to have an excellent safety profile and protracted transgene expression (Seiler et al., 2007; Brunetti-Pierri and Ng, 2008). Other advantages of HDAd are a large cloning capacity (up to 37 kb) and a greatly attenuated adaptive host immune response (Muruve et al., 2004). Thus, in specific embodiments the invention concerns HDAd as the backbone for the targeted vector for DRG neuron delivery.
The cellular tropism of Ad serotype 5 (Ad5) is determined by the cell-surface expression of primary attachment sites, the Coxsackie and Adenovirus receptor (CAR) (Bergelson et al., 1997; Tomko et al., 1997) and heparan sulfate proteoglycans (HSPG) (Dechecchi et al., 2001) and/or low-density lipoprotein receptor-related protein, and via the integrins, which act as secondary internalization receptors (Shayakhmetov et al., 2005; Koizumi et al., 2006; Parker et al., 2006). Cells deficient in the expression of these receptors are transduced at a greatly reduced efficiency. Little or no CAR is expressed by DRG neurons, which are poor targets for unmodified Ads (Hotta et al., 2003). Considerable effort has been expended on the generation of first generation Ads that target normally refractory tissues, as well as their retargeting to alternative cell-type specific receptors. Three major strategies have been employed in this respect: genetic modification of Ad capsid proteins, conjugation of adaptor proteins such as antibody, or bispecific fusion proteins, and chemical modification by polymers with targeting ligands (Waehler et al., 2007; Mizuguchi and Hayakawa, 2004). Genetic modification appears to the most popular of these approaches. This technology is particularly appealing for HDAd because capsid proteins of HDAd are supplied by the helper virus (HV). Armed with a library of targeting HVs, one can produce HDAds that potentially target any tissue by applying the specific HV at the final amplification. To date, despite the many potential advantages of HDAd, targeting HDAd by genetic modification has been limited to the introduction of peptide ligands into the HI loop (Biermann et al., 2001) or replacement of fiber gene with that of a different Ad serotype (Wang et al., 2005).